Biological cell and tissue fixation by laser irradiation

ABSTRACT

Systems and methods of fixing biological cells by laser irradiation. A method according to one embodiment of the present invention includes positioning a sample (of the cell or tissue) in a light pathway of a fixation source. The fixation source configured to emit electromagnetic radiation having a wavelength along the light pathway. The sample is exposed to the electromagnetic radiation for an exposure time.

RIGHTS OF THE GOVERNMENT

The invention described herein may be manufactured and used by or forthe Government of the United States for all governmental purposeswithout the payment of any royalty.

Pursuant to 37 C.F.R. § 1.78(a)(4), this application claims the benefitof and priority to prior filed co-pending Provisional application Ser.No. 14/670,06 (pending), filed Mar. 26, 2015, which is expresslyincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to the field of fixation andpreservation of biological specimens. More particularly, it relates tomethods and systems for fixing and preserving a biological specimenusing optical irradiation.

BACKGROUND OF THE INVENTION

The purpose of fixing biological specimens is to preserve the sample asclose to its natural state as possible so that it may be examined later.Conventional fixation methods typically use physical means such as heator freeze-drying, or chemical means, such as aldehydes (e.g.,formaldehyde, paraformaldehyde, or glutaraldehyde) or alcohols (e.g.,ethanol or methanol) to preserve a biological specimen for furtherpreparation and examination. In heat fixation, a slide with a biologicalspecimen is passed through a flame to adhere the specimen to the slide.Other methods of heat fixation include exposing the sample to microwaves(with or without the presence of chemical fixatives in the specimen).Samples may also be fixed with cold temperatures by freeze drying. Inchemical fixation, the sample is immersed in the fixative thatpenetrates the specimen to preserve the sample. Many chemical fixatives,such as aldehydes, act to crosslink the proteins in the cells ortissues. Precipitating fixatives, such as alcohols, cause the proteinsin the sample to precipitate or aggregate by reducing the solubility ofthe proteins or disrupting their tertiary structure.

These conventional methods suffer from a variety of limitations.Importantly, fixation using one or more of these current methods maydisrupt the structure and composition of the cellular components,creating fixed samples that do not truly reflect the properties of theliving cells or tissues. Conventional fixation methods often takeseveral hours, meaning that the sample structure and/or properties maybe altered while the fixation is occurring. Fixation may distort oralter the sample due to, for example, shrinkage upon dehydration of thesample or swelling/shrinkage upon placement of the specimen in anon-aqueous chemical fixative. Some components of the specimen (e.g.,lipids) may also be lost during the fixation process. In addition, theconventional methods typically require that the entire sample containedin the sample container (e.g., slide, dish, etc.) be fixed at once.Furthermore, almost all fixation chemicals are extremely hazardous andspecial precautions must be taken to avoid accidental and/or chronicoverexposure. The hazards associated with these chemicals range frominhalation and skin/eye contact hazards to severe flammability.

SUMMARY OF THE INVENTION

The present invention overcomes the foregoing problems and othershortcomings, drawbacks, and challenges of conventional fixationprocesses. While the invention will be described in connection withcertain embodiments, it will be understood that the invention is notlimited to these embodiments. To the contrary, this invention includesall alternatives, modifications, and equivalents as may be includedwithin the spirit and scope of the present invention.

According to one embodiment of the present invention, a method of fixinga sample includes positioning a sample in a light pathway of a fixationsource. The fixation source configured to emit electromagnetic radiationhaving a wavelength along the light pathway. The sample is exposed tothe electromagnetic radiation for an exposure time.

In accordance with another embodiment of the present invention, a methodof fixing a sample includes positioning a sample in a light pathway of afixation source. The fixation source configured to emit electromagneticradiation having a wavelength along the light pathway. A first portionof the sample is exposed to the electromagnetic radiation for a firstexposure time, and a second portion of the sample is exposed to theelectromagnetic radiation for a second exposure time.

Yet another embodiment of the present invention includes a system forfixation of a biological sample. The system comprises a microscope, afixation source, and beam delivery-focusing optics. The microscope isconfigured to visualize at least a portion of the biological sample. Thefixation source is configured to emit an electromagnetic radiationhaving a wavelength and operably coupled to the microscope so as toirradiate the portion of the biological sample. And the beamdelivery-focusing optics, which is positioned between the fixationsource and the sample, is configured to adjust at least one parameter ofthe electromagnetic radiation.

Additional objects, advantages, and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art upon examination of thefollowing or may be learned by practice of the invention. The objectsand advantages of the invention may be realized and attained by means ofthe instrumentalities and combinations particularly pointed out in theappended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate embodiments of the presentinvention and, together with a general description of the inventiongiven above, and the detailed description of the embodiments givenbelow, serve to explain the principles of the present invention.

FIG. 1 is a side view of a first exemplary system according to thepresent invention comprising an optical microscope.

FIG. 2 is a side view of a second exemplary system according to thepresent invention comprising a fluorescence microscope.

FIG. 3 is a flowchart illustrating a method of using the exemplarysystem of FIG. 1 in accordance with an embodiment of the presentinvention.

It should be understood that the appended drawings are not necessarilyto scale, presenting a somewhat simplified representation of variousfeatures illustrative of the basic principles of the invention. Thespecific design features of the sequence of operations as disclosedherein, including, for example, specific dimensions, orientations,locations, and shapes of various illustrated components, will bedetermined in part by the particular intended application and useenvironment. Certain features of the illustrated embodiments have beenenlarged or distorted relative to others to facilitate visualization andclear understanding. In particular, thin features may be thickened, forexample, for clarity or illustration.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes methods and systems for the histologicalfixation and preservation of biological specimens (cells or tissues) byacute exposure to electromagnetic radiation, including visible,ultraviolet, and infrared radiation. In some embodiments, theelectromagnetic radiation comprises wavelengths ranging from about 100nm to about 7,000 nm. Exposure of the biological specimen to acontrolled dosage of electromagnetic irradiation preserves the materialin its existing state so that it may be prepared for further examinationat a later time. The presently disclosed methods and systems may be usedto preserve samples for observation by techniques, such as opticaltechniques, tunneling electron microscopy (TEM), scanning electronmicroscopy (SEM), and atomic force microscopy (AFM), as well as tostabilize specimens for transportation. Biological fixation according tothe present invention allows for a single region of interest ofadjustable size to be fixed, while leaving the remainder of the cellculture or tissue viable for further examination and experimentation.This ability to achieve very controlled and limited fixation allows formulti-time point studies, potentially within in the same sample orspecimen.

In addition, methods and systems according to the present inventionachieve rapid fixation to reduce or eliminate many of the physiologicalor chemical changes in the sample that are commonly observed with otherfixation processes due in part to the duration of the procedure. Becauseembodiments of the present invention achieves fixation in seconds tominutes—as opposed to hours or even days with conventional methods—asnapshot of a continuously changing microenvironment may be captured toprovide optically fixed samples that may then be observed formorphological and chemical characteristics, as well as time-dependentchanges within the same sample. The presently disclosed methods offixation may also increase the mechanical stability or strength of thesample material to maintain the morphology of the sample. Properfixation is particularly important for immunohisto-chemical orimmunofluorescence methods that visualize molecular markers in cellsusing antibodies. Because of the size of the antibodies, use of theseapproaches with living cells is usually extremely difficult; as such,these methods typically employ fixed cells that have been madepermeable. The presently disclosed methods and systems provide fixationprocedures that may help to retain a level of reactivity to theantibodies and nucleic acid probes that more closely mimics livingcells.

Referring to the drawings, wherein like reference numerals may designatelike or corresponding parts throughout the several views, FIG. 1 is aside view of an exemplary system according to one embodiment of thepresent invention. The illustrated embodiment includes an opticalmicroscope 100, wherein some components of the optical microscope 100are omitted for clarity and to better illustrate certain aspects of theinvention. The optical microscope 100 comprises a light source 105 andat least one condenser lens 110. The light source 105 is configured togenerate light which illuminates a biological sample 112 on or in asample container (illustrated here as a glass slide 113) on a samplestage 115. Examples of suitable systems may include, for example, aninverted or upright microscope, an inspection microscope, a telescope,or any other suitable combination of a lens system, either through anoptical fiber or directly from the laser.

The biological sample 112 may be any cellular and tissue specimenobtained from in vivo and in vitro experiments and may include, forexample, cells obtained by cell culture methods, a cell smear, cellsisolated from a tissue section, or a tissue section. Cellular specimensmay be stained, labeled, frozen, or otherwise treated and may beadherent or in suspension. The sample container 113 will vary based onthe type of biological sample 112 and may comprise, for example, amicroscope slide (such as the illustrated glass slide 113) with orwithout coverslip, a dish, a well plate, a petri dish, a cuvette, acapillary tube, a syringe, or any other suitable container configured tohold biological samples 112. The sample container 113 may compriseglass, plastic, or other suitable transparent material and may be areflective or somewhat reflective to the electromagnetic radiationgenerated by the fixation source but also configured to transmit lightfrom the light source 105. The sample container 113 may also be asubstrate used for techniques such as AFM, TEM, and SEM. Thesesubstrates include, but are not limited to, mica, silicon, gold coatedsubstrates, silicon nitride, crystals, quartz, and sapphire.

Referring again to FIG. 1, the light source 105 may include anelectronic shutter 145 to control illumination of the biological sample112 through the glass slide 113. The light emitted from the light source105 (illustrated as beam 118 and defining a light pathway) andtransmitted through the biological sample 112 on the glass slide 113 istransmitted to a microscope objective 120, which may contain one or moreobjective lenses and which is coupled to an eyepiece 125. The eyepiece125 permits a user to observe a magnified view of the biological sample112.

The optical microscope 100 according to the illustrated embodiment ofFIG. 1 further comprises a fixation source 130 and a beamdelivery-focusing optics (enclosed in dashed circle 133) comprising oneor more lenses 135, one or more mirrors 140, or combinations thereof andis configured to deliver electromagnetic radiation from the fixationsource 130 to the biological sample 112.

The fixation source 130 may comprise, for example, a laser, a lightemitting diode, lamp, bulb, or other suitable source configured toproduce one or more of visible light, ultraviolet light, or infraredirradiation (illustrated as beam 132 for the light pathway), and may befurther configured to generate single wavelength, multiple wavelengths,continuous wave, quasi-continuous wave, or pulsed waves. In someembodiments of present invention, the fixation source 130 deliversvisible, ultraviolet, and/or infrared radiation having wavelengthsranging from about 100 nm to about 7,000 nm. One of ordinary skill willunderstand that the components of the beam delivery-focusing optics 133will be specific to each setup and may comprise, other than the lenses135 and mirrors 140, one or more of optical filters, beam splitters,beam combiners, mirrors, and fiber cables. An electronic optical shutter(not labeled) may be placed in the optical path of the fixation source130 to control the duration or exposure of the irradiation on thebiological sample 112.

In the embodiment depicted in FIG. 1, the fixation source 130 ispositioned external to the microscope 100 and between the microscopeobjective 120 and the biological sample 112 on the glass slide 113;however, such arrangement is not limiting and may be located at anysuitable position within or without the optical microscope 100. Forexample, although not specifically shown, the fixation source 130 may bepositioned within the microscope 100 and between the eyepiece 125 andthe microscope objective 120 such that the electromagnetic radiationpasses through the microscope objective 120 prior to striking thesample. Thus, a size of the light spot generated by the laser may befixed to a specific diameter or changed by varying objectives or lenses.According to still other embodiment, not specifically illustratedherein, the fixation source 130 may be positioned on a side of theeyepiece 125 that opposes the objective 120 and the size of the lightspot may be altered by using a telescope or other such beam expansionbetween the fixation source 130 and the eyepiece 125 and prior to themicroscope.

It will be readily apparent to one skilled in the art having the benefitof the disclosure herein that illustrated embodiment of FIG. 1 may bepreferable in instances where observation and positioning of thebiological sample 112 is needed prior to or during fixation.

Referring now to FIG. 2, wherein a side view of another exemplary systemaccording to the present invention comprising a fluorescence microscope200 is shown, a light housing 250 configured to contain both a lightsource and a fixation source (corresponding to the light source 105 andthe fixation source 130, respectively, of FIG. 1) of within a singlehousing is shown. The light source may comprise, for example, a mercuryor xenon lamp. Light (illustrated as beam 262), whether generated fromthe light source or the fixation source within the housing 250 istransmitted through one or more excitation filters 255. A beam splitter260, which may comprise, for example, a dichroic mirror, reflects aportion of the light beam 262 transmitted through the filters 255 towardthe microscope objective 120 onto a biological sample 112 on the glassslide 113. The light 262 may be absorbed by or reflected by or at thebiological sample 112. In those instances when light 262 is absorbed bythe biological sample 112 (or a dye or stain applied thereto), light ofa different wavelength may be emitted (for example, fluorescent light)and is directed (illustrated as beam 268) away from the biologicalsample 112, through the beam splitter 260, to and through an emissionfilter 265, and, ultimately, toward the eyepiece 125 and user (notshown). Light reflected by the biological sample 112 or the glass slide113 (or the stage 115) is also directed along the direction of beam 268.

It will be readily apparent to one skilled in the art having the benefitof the disclosure herein that illustrated embodiment of FIG. 2 may alsobe useful in instances where observation and positioning of thebiological sample 112 is needed prior to or during fixation. However,one benefit to the illustrated embodiment of FIG. 2 is that the user(not shown) may optionally switch between operation of the light source,the fixation source, or both at the housing 250.

Turning now to FIG. 3 (with reference again to FIG. 1) a method of usingembodiments of the present invention is shown. At start, the biologicalsample 112 is positioned with the glass slide 113 on the stage 115 suchthat a desired region or portion of the sample 112 to be fixed islocated within a field of view or exposure area of the fixation source130 (Block 310). Some embodiments of the method include, optionally, anadditional treatment and/or manipulation of the biological sample 112,such as delivery of a pharmaceutical or probe or exposure of the sampleto an experimental variable (Block 320). One or more parameters of thefixation source 130 and/or the beam delivery-focusing optics 133 may beadjusted (Block 330) as needed to achieve, for example, a desiredwavelength, power, spot size, and/or distance between the fixationsource 130 and the biological sample 112.

A portion of the biological sample is fixed 112 by exposing thebiological sample 112 to electromagnetic radiation emitted from thefixation source 130. In that regard, the optical shutter (not shown) onthe fixation source 130 is opened for a brief period of time (typicallyranging from a few microseconds to a few minutes) in order to deliver apredetermined dosage of electromagnetic radiation having a specifiedwavelength to the biological sample 112. The dosage (generally measuredin J/cm²) may be calculated by multiplying the intensity or irradianceof the electromagnetic radiation (generally measured in W/cm²) by theexposure time (generally measured in seconds or minutes).

Following fixation (Block 340), a determination is made whetheradditional fixation and/or treatment/manipulation of the biologicalsample 112 will occur (Decision Block 350). Where no additional handlingof the biological sample 112 is desired (“No” branch of Decision Block350), the biological sample 112 is removed (Block 360) and the methodterminates. Where additional fixation and/or treatment/manipulation ofthe sample is desired (“Yes” branch of Decision Block 350), the methodreturns to further adjust one or more parameters of the fixation source130 and/or the beam delivery-focusing optics 133 (Block 330) andcontinues until the desired number of iterations of additional fixationand/or treatment/manipulation of the biological sample 112 are achieved.

In some embodiments, the biological sample 112 and/or the samplecontainer 113 may be repositioned one or more times to define a newfield of view or exposure area. In other embodiments, additionaltreatment/manipulation of the biological sample 112 may be performedbefore additional fixation(s). In further embodiments, the repeatedrepositioning and/or treatment/manipulation of the biological sample 112(in Block 320) and fixation (in Block 340) may be implemented as aroutine into microscope software so that a “fixation” protocol may beselected to automate the fixation routine (such as using one or morestepper motors to manipulate the stage 105). With all treatmentscomplete, the biological sample 112 is removed (step 360), and themethod terminates.

In some embodiments, the biological sample 112 may still optionallyundergo a final treatment (Block 370) to prepare the biological sample112, for example, for additional post-fixation examination,transportation, and/or storage. This final treatment may include, forexample, labeling with tagged antibodies specific to desired antigens orother labeling or staining techniques. By fixing multiple samples on asingle slide, the presently disclosed method further allows for thesedifferent samples to be screened simultaneously with many differentantibodies.

The following examples illustrate particular properties and advantagesof some of the embodiments of the present invention. Furthermore, theseare examples of reduction to practice of the present invention andconfirmation that the principles described in the present invention aretherefore valid but should not be construed as in any way limiting thescope of the invention.

Example

Chinese hamster ovarian (“CHO-K1”) cells were acquired from AmericanType Culture Collection (ATCC, Manassas, Va.) and cultured according tothe supplier's protocol at 37° C. in 5% CO₂ and 95% humidity. F-12KMedium was supplemented with 10% FBS with 1 I.U./mL penicillin and 0.1μg/mL streptomycin antibiotics. For experimentation, cells were grown on35 mm glass-bottom-coverslip dishes that were demarcated into separateexposure areas. To prepare cells for labeling, cell media was removed byvacuum aspiration, the cells were rinsed twice with a buffered salinesolution, and fresh buffer was added.

The cells were treated in accordance with embodiments of the presentinvention and using a fixation source consisting of a 488 nm argon laserpositioned such that its beam is was routed through the epi-illuminationpathway of an inverted microscope and through an objective lens. Priorto the microscope, the beam was expanded to fill an illuminationdiameter of about 150 μm at the sample plane. Laser exposures of 10 mW,20 mW, and 50 mW were applied to the cells (about 50 W/cm², 100 W/cm²,and 300 W/cm², respectively) for a test time period. After eachexposure, the next area was selected and the cells were exposed to newtest parameters.

To test whether the cells were fixed, the dishes were rinsed repeatedlywith phosphate buffered saline solution to remove any non-fixed cellsand then labeled with a fixed cell labeling kit to visualize thecellular structures. Exposures of 10 seconds, 30 seconds, 1 minute, 2minutes, and 3 minutes at 10 mW or 20 mW all showed some level of cellfixation within the exposed areas. Exposures times of 2 minutes orlonger showed consistent fixation at 10 mW. Exposures of 1 minute, 2minutes, and 3 minutes with 50 mW all resulted in cell fixation (10seconds and 30 seconds were not tested). Unexposed areas of the cellplate did not show any cells or fluorescence.

As provided herein, the present invention may be implemented as anintegral part of a microscope of other suitable system or as anextension or modular add-on for an existing systems. The system mayallow for light microscope observation of surrounding cells and tissuein a living state while simultaneously fixing cells at certain timepoints.

Because the methods and systems describe herein do not require the useof hazardous and/or flammable chemicals or the use of proper protectiveequipment (e.g., fume hoods, respirators, gloves, to name a few), it mayalso be used as part of a portable system to allow for fixation and/orinactivation in remote or flexible locations. In addition, fixation ofsamples using embodiments of the present invention may be performed by alayperson without formal training, reducing the number of specialistsrequired at remote locations. The fixed samples and/or images of thefixed samples may then be sent to a central location for analysis orfurther preparation to detect or diagnose, for example, exposure to abiological agent. The ability to quickly preserve and safely maintainsamples during transport is desirable to professionals in a variety offields ranging from health professionals working in hazardousenvironments to research scientists collecting and preserving samples inthe field. Safe and rapid preservation and inactivation of samplescontaining possible biological contaminants is particularly desirable toreduce the risk of outbreaks and increase the likelihood of properdiagnosis.

While the present invention has been illustrated by a description of oneor more embodiments thereof and while these embodiments have beendescribed in considerable detail, they are not intended to restrict orin any way limit the scope of the appended claims to such detail.Additional advantages and modifications will readily appear to thoseskilled in the art. The invention in its broader aspects is thereforenot limited to the specific details, representative apparatus andmethod, and illustrative examples shown and described. Accordingly,departures may be made from such details without departing from thescope of the general inventive concept.

What is claimed is:
 1. A system for fixation of a biological samplecomprising: a microscope configured to visualize first and secondportions of the biological sample; a fixation source configured to emitan electromagnetic radiation having a wavelength and operably coupled tothe microscope so as to irradiate the first and second portions of thebiological sample; a moveable stage configured to support the biologicalsample with respect to the microscope and to move from the first portionof the biological sample to the second portion the biological sample;and beam delivery-focusing optics between the fixation source and thebiological sample and configured to adjust at least one parameter of theelectromagnetic radiation, wherein the first portion of the biologicalsample may be exposed to the electromagnetic radiation for a firstexposure time such that the first portion is fixed and, at some timeafter the first portion is fixed, the second portion of the biologicalsample may be exposed to the electromagnetic radiation for a secondexposure time such that the second portion is fixed.
 2. The system ofclaim 1, wherein the fixation source is external to the microscope. 3.The system of claim 1, wherein the fixation source is within themicroscope.
 4. The system of claim 1, wherein the fixation sourcefurther comprises: a shutter configured to adjust the first exposuretime, the second exposure time, or both.
 5. The system of claim 1,wherein wavelength of the electromagnetic radiation ranges from about100 nm to about 7,000 nm.
 6. The system of claim of claim 1, wherein thebiological sample is positioned on a glass slide on the moveable stage.